The roles of non-coding RNAs in the self-renewal and differentiation of pluripotent stem cells

The roles of non-coding RNAs in the self-renewal and differentiation of pluripotent stem cells

Funding Type: 
New Faculty II
Grant Number: 
RN2-00923
Award Value: 
$1,406,823
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a novel miRNA, Esdmir-1, whose loss-of-function significantly promotes the reprogramming of iPS cells. This is an important finding, not only does it set up a paradigm for our future studies, it also provides an attractive methodology to improve iPS reprogramming in human. Our future effort in the next funding period (year 2) will be focused on completing the studies on Esdmir-1, evaluating the functions of additional candidate miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.

Year 2

The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a family of novel miRNAs that promote differentiation in pluripotent stem cells. This is an important finding because we demonstrated that repression of these miRNAs significantly enhances reprogramming efficiency. miRNAs functions can be modulated by transient transfection of oligonucleotide based antagonist, therefore, our findings are likely to lead to an approach to greatly promote iPSC generation in clinical application. Our future effort in the next funding period (year 3) will be focused on functional characterizations of additional miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.

Year 3

So far, our CIRM funded project have provided strong evidence suggesting miRNAs as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells, thus playing an important role in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for year 3. In short, we have carefully characterized the roles of miR-34 miRNAs in somatic reprogramming and indicated the importance of miR-34 inhibition in promoting stem cell self-renewal and somatic reprogramming. In addition, we completed two functional screens to identify miRNAs whose overexpression or inhibition regulates ES cell self-renewal and ES cell differentiation. Functional validation has been performed for majority of the hits from the screen. In particular, we have identified a miRNA, miR-meso, which specifically promotes mesoderm differentiation and represses ectoderm differentiation. Finally, using recombineering technology, we have constructed a BAC with Flag-tagged Lin28 at its endogenous locus. We have also constructed a piggy bac vector that allows us to generate stably integrated ES cell line that expresses Flag-tagged LIN28 for identification of novel LIN28 bound non-coding RNAs. These progresses significantly improved our understanding on the roles of non-coding RNAs in regulating stem cell self-renewal and pluripotency, and may lead to novel strategy for generating completely pluripotent human stem cells for clinical applications. In 2011, a portion of our results funded by the CIRM project were published in Nature Cell Biology as a cover story, and we also filed a patent application reporting a novel strategy to increase somatic reprogramming efficiency.

Year 4

Our CIRM funded project have provided strong evidence suggesting microRNAs (miRNA), and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Although non-coding RNAs, including miRNAs, do not have protein coding capacity, but they possess strong effects in regulating Using genomic approaches, embryonic stem cell culture and induced pluripotent stem cell culture and molecular biology, we were able to screen for, identify and characterize a number of non-coding RNAs, particularly, microRNAs, as important regulators for stem cell self-renewal and differentiation.Our results can have profound implications on the role of ncRNAs in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for the year 4 of our CIRM funded project. . Last year, we have successfully carried out a functional screen to identify miRNAs with important roles in regulating ES cell self-renewal. Among the miRNA candidates emerged from this screen, we primarily focused on the functional and mechanistic characterization of several important miRNA regulators for ES cell pluripotency. We investigated roles of miR-34 miRNAs, whose deficiency significantly promotes somatic reprogramming. We studied the role of miR-34 in the epigenetic remodeling during somatic reprogramming. We also studied the function of mir-290-295 cluster, which constitute the majority of miRNA species in the ES cells. We generated ES cell deficient for the mir-290-295 miRNA cluster to characterize its effects on ES cell self-renewal, and we also explored the functional redundancy of the mir-290-295 family miRNAs by functionally characterized related miRNA family, including the mir17-92 miRNAs. In the previous funding period, we also carried out a screen to identify miRNAs or non-coding RNA with important roles in regulating ES cell differentiation. Here, we functionally characterized the effects of miR-meso in mesoderm differentiation both in vitro and in vivo, using teratoma assays and KO ES cells. In addition, we identified a long ncRNA that exhibit an interesting pattern of expression during ES cell differentiation. We characterized its unique expression alteration during ES cell differentiation, and we are currently generating the knockout ES cells for this long ncRNA to further investigate its function. Finally, we characterized miRNA expression profiles during ES cell to Epiblast stem cell differentiation, and identified additional candidate miRNAs that regulate the exit of pluripotency of ES cells during their differentiation. Finally, we successfully established the biological system to generate human iPSCs from human dermal fibroblasts, and will use this system to explore the role of specific ncRNAs in human somatic reprogramming. Our previous results using mouse ES cells and iPSCs have prepared us well in exploring the significance of our finding in human. This will be a main focus to achieve in our last funding period.

Year 5

Our CIRM funded project have provided strong evidence suggesting miRNAs, and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Consistently, these ncRNAs play an important role in the generation of induced pluripotent stem cells (iPSCs). Using expression studies, functional screening and candidate approaches, we have identified and characterized the roles of multiple miRNAs and long ncRNAs (lncRNAs) in regulating the self-renewal and differentiation of pluripotent stem cells. These non-coding RNAs are essential component of a regulatory network that regulate stem cell cell fate potential, self-renewal and differentiation potential.

© 2013 California Institute for Regenerative Medicine